Acrylamide is a colourless and odourless crystalline solid that is an important industrial monomer commonly used as a cement binder and in the synthesis of polymers and gels. Based on various in vivo and in vitro studies there is clear evidence on the carcinogenic and genotoxic effects of acrylamide and its metabolite glycidamide (Wilson et al, 2006; Rice, 2005). Acrylamide was evaluated by the International Agency for Research on Cancer (IARC) in 1994 and it was classified as “probably carcinogenic to humans” on the basis of the positive bioassays completed in mice and rats, supported by evidence that acrylamide is bio-transformed in mammalian tissues to the genotoxic glycidamide metabolite (IARC, 1994). The biotransformation of acrylamide to glycidamide is known to occur efficiently in both human and rodent tissues (Rice, 2005). In addition to the IARC classification, ‘The Scientific Committee on Toxicity, Ecotoxicity and the Environment’ of the European Union and the independent ‘Committee on Carcinogenicity of Chemicals in Food, Consumer Products and the Environment’ in the UK, both advised that the exposure of acrylamide to humans should be controlled to a level as low as possible due to its inherently toxic properties including neurotoxicity and genotoxicity to both somatic and germ cells, carcinogenicity and reproductive toxicity.
With respect to human epidemiological studies on dietary acrylamide exposure, there is no evidence for any carcinogenic effect of this chemical; however, it is also recognized that these epidemiological studies on acrylamide may not be sufficiently sensitive to reveal potential tumours in humans exposed to acrylamide (Rice, 2005; Wilson et al, 2006).
In 2002, the Swedish National Food Authority published a report detailing the concentrations of acrylamide found in a number of common foods, specifically heat-treated carbohydrate-rich foods such as French fries and potato chips. The list has now been expanded to include grain-based foods, vegetable-based foods, legume-based foods, beverages such as coffee or coffee substitutes; Table 1 shows FDA data on acrylamide concentrations in a variety of Foods.
It is now established that acrylamide is formed during the cooking of foods principally by the Maillard reaction between the amino acid asparagine and reducing sugars such as glucose, with asparagine being the limiting precursor (Amrein et al, 2004; Becalski et al 2003; Mustafa et al 2005; Surdyk et al, 2004; Yaylayan et al 2003).
There have also been a number of approaches attempted to reduce acrylamide content in food including the addition of commercial preparations of the enzyme asparaginase (Acrylaway®, Novozymes, Denmark and PreventASe, DSM, Netherlands), extensive yeast fermentation for 6 hours (Fredriksson et al, 2004), applying glycine to dough prior to fermentation (Brathen et al, 2005; Fink et al 2006), dipping potatoes into calcium chloride prior to frying (Gokmen and Senyuva, 2007), replacing reducing sugars with sucrose (Amrein et al, 2004), general optimization of the processing conditions such as temperature, pH and water content (Claus et al, 2007; Gokmen et al, 2007) and studies regarding different choices of raw materials (Claus et al, 2006). All of these listed approaches are inadequate to some degree or have inherent issues that make them impractical during the manufacture of food products including cost, effect on organoleptic properties of the food and/or ineffective acrylamide reduction under food processing conditions.
Like many microorganisms, Saccharomyces cerevisiae is capable of naturally consuming/degrading the acrylamide precursors asparagine and reducing sugars. This may be the reason for an observed reduction of acrylamide content in bread after an extensive fermentation time of 6 hours (Fredriksson et al, 2004). However, such an extensive fermentation time to effectively reduce acrylamide is impractical in modern food production processes.
In S. cerevisiae, the genes responsible for asparagine degradation are ASP1 and ASP3 that encode for a cytosolic asparaginase and a cell-wall asparaginase, respectively. There are also at least 41 genes in S. cerevisiae annotated to the term ‘amino acid transport’ and six of these transporters are known to be capable of transporting asparagine into the cell [“Saccharomyces Genome Database” http://www.yeastgenome.org/(Oct. 1, 2009)]. The gene names for these six asparagine transporters in S. cerevisiae are GAP1, AGP1, GNP1, DIP5, AGP2 and AGP3. It is also well established that S. cerevisiae is able to use a wide variety of nitrogen sources for growth and that in mixed substrate cultures it will sequentially select good to poor nitrogen sources (Cooper, 1982). This sequential use is controlled by molecular mechanisms consisting of a sensing system and a transcriptional regulatory mechanism known as nitrogen catabolite repression (NCR). In general, NCR refers to the difference in gene expression of permeases and catabolic enzymes required to degrade nitrogen sources. The expression of nitrogen catabolite pathways are regulated by four regulators known as Gln3p, Gat1p, Dal80p and Gzf3p that bind to the upstream activating consensus sequence 5′-GATAA-3′. Gln3p and Gat1p act positively on gene expression whereas Dal80p and Gzf3p act negatively. In the presence of a good nitrogen source, Gln3p and Gat1p are phosphorylated by the TOR kinases Tor1p and Tor2p; then form cytosolic complexes with Ure2p and are thereby inhibited from activating NCR-sensitive transcription. In the presence of poor nitrogen sources or nitrogen starvation Gln3p and Gat1p become dephosphorylated, dissociate from Ure2p, accumulate in the nucleus and activate NCR-sensitive transcription.
It is also well documented that a particular mutation of URE2 yields a dominant mutation referred to as [URE3]. [URE3] is a yeast prion that is formed by the autocatalytic conversion of Ure2p into infectious, protease-resistant amyloid fibrils (Wickner, 1994). The phenotypes of S. cerevisiae cells lacking a functional Ure2p and [URE3] infected cells are similar as they no longer respond to NCR (Wickner, 1994; Wickner et al, 1995). As noted above, in response to a good nitrogen source, Ure2p is involved in the down-regulation of Gln3p and Gat1p activity.